Bone marrow transplantation has emerged as a major therapeutic modality for immunodeficiencies and blood cancers. The success of this therapy depends on matching major histocompatibility complex (MHC) alloantigens and on immunosuppression. Despite these preventive measures, immunological rejection ensues because minor histocompatibility (minor H) antigen incompatibilities lead to graft versus host disease. Based on the hypotheses that immune responses to minor H antigens are mediated by cytotoxic T lymphocytes (CTL) when they are presented as self peptides by MHC class I molecules and that multiple minor H incompatibilities lead to CTL responses to dominant alloantigens as well as the fact that the molecular nature and the basis of immunodominance of these alloantigens are not known, this proposal is focused on two questions: 1. What is the molecular nature of the immunodominant minor H antigenic peptide(s) that elicits C57BL/10 anti-BALB.B response? and 2. What is the molecular basis o immunodominance? A rational approach to delineate the molecular identity of minor H antigens would be to isolate self peptides from class I molecules and to identify the minor H peptide using specific CTLs as probes followed by amino acid sequence analysis of the biologically active peptide(s). Obtaining the active peptide in sufficient amounts and in adequately pure form for sequence analysis is a major limitation to this approach because a given class I molecule constitutively binds and presents over 10,000 different self peptides. To overcome this problem self peptides will be isolated from 3--5 mg of genetically overexpressed soluble H-2Kb, purified by reversed-phase (RP) HPLC and scanned with minor H antigen specific CTLs derived from C57BL/10 mice against BALB.B (both H-2b but multiple minor H disparate). Since the active RP-HPLC fraction will contain a mixture of peptides, it will be purified further using different mobile and/or stationary phases. The final active fraction will be sequenced, the corresponding peptide synthesized, and its ability to sensitize targets for minor H specific CTL will be determined. To test whether antigen competition at the level of minor H peptide presentation is the basis of immunodominance, the amount of immunodominant epitope(s) will be quantitated from the sequence data and its affinity for Kb will be determined. As a part of the long term goals of this laboratory, we will use the results from the above studies to define peptide antagonists, either synthetic or naturally occurring among self peptides, to overcome allograft rejection. Further, the above studies also will provide insights on the minimum variability required in a, normally, self peptide to turn into a non-self autoantigen and how tumor cells evade immunesurveillance.